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  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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  • C07K14/4747—Apoptosis related proteins
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  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
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  • C07K14/82—Translation products from oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/028—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a herpesvirus

Definitions

• the present invention relates to the identification of genes involved in the molecular pathways of tumor suppression and the use of the genes thus identified for the treatment of certain gene dysfunctions, in particular cancers.
• the present invention was made possible by the isolation of cDNAs corresponding to messenger RNAs expressed or repressed during the process of apoptosis induced by the suppressor gene p53.
• oncogenes due to their deregulation in cancer (most often resulting from a mutation or translocation) will induce a positive signal which will promote neoplastic growth.
• suppressor genes due to their deletion, the absence of their expression by mutation of the promoter, for example, or even mutations which will modify the structure and function of the protein, will be incapable in cancer of providing the signal which, normally, should slow down this abnormal growth. As a result, dysfunction in suppressor genes contributes to neoplastic transformation.
• the object of the present invention is the isolation of genes normally having an action in tumor suppression and of which it will then be possible to monitor and treat any dysfunctions.
• the isolation of these genes makes it possible to have recourse to gene replacement therapy or else to the synthesis of pharmacological agents, protein or non-protein, which, directly or indirectly, through their action on the promoters, will induce the activation and expression of these genes, or even the synthesis of pharmacological agents which will make it possible to mimic the physiological effect of these suppressor genes.
• the final objective is either to inhibit tumor growth, or better, to induce the apoptotic process of these tumor cells, that is to say to lead the tumor cells to "commit suicide".
• the present invention relates to the detection of genes which are involved in this apoptosis. Indeed, each cell has within it a physiological death program. It is also a physiological process which is involved in development in order to maintain the homeostasis of the body and not to see abnormal cell proliferations being established, even if, moreover, they have no character. clever.
• mice nullizygous for p53 were much more sensitive to the formation of tumors. It has also been demonstrated that, in cancers, the p53 gene is very often altered and leads to the production of proteins incapable of conveying the message of apoptosis.
• the present invention is based on the observation that it is not possible, or at least that it seems very difficult, to set up a direct substitution therapy during a dysfunction of the p53 gene. Indeed, p53 mutates as it is in cancer will cancel the physiological effect of normal p53.
• the present invention is therefore devoted to studying the genes located downstream of p53 in order to "bypass" the difficulty mentioned above.
• the isolation of genes involved in suppression has been carried out either by positional cloning or by the use of double hybrids.
• the first method made it possible, by statistical calculation, to calculate the highest probability or could locate, at the chromosomal level, a candidate suppressor gene for a very specific type of cancer, especially those of familial origins.
• the double hybrid system allows the proteins that interact with a given gene to be isolated one by one.
• sequences are sequences whose function is known and which are involved in the process of apoptosis induced by the suppressor gene p53.
• this method has been used on a cell model described by Moshe Oren, these are mouse tumor cells which have been transfected with a stable mutant of the p53 gene.
• the expression of this gene is thermosensitive, that is to say that under cell culture conditions at 37 ° C. the protein produced is a mutated protein, that is to say that it cannot play the role of tumor suppressor and therefore that the corresponding cell line develops in the form of a malignant cell, while at the temperature of 32 ° C. the expressed p53 protein, like the natural protein, is capable of playing the role of suppressor and prevents the corresponding cell line from becoming malignant.
• the present invention firstly relates to a nucleotide sequence corresponding to a gene comprising:
• the present invention relates to a human gene involved in the suppression cascade induced by p53 as well as the use of the sequences of this gene, both at the diagnostic level and at the therapy level, as well as for carrying out models intended to test anti-cancer products as well as their application as an antiviral agent.
• the present invention therefore also relates to a nucleotide sequence corresponding to a gene comprising:
• nucleotide sequences corresponding to the entire gene and fragments of this gene in particular when they code for an equivalent protein as will be described below.
• the nucleotide sequences can be either DNA or RNA or sequences in which some of the nucleotides are unnatural, either to improve their pharmacological properties or to allow their identification.
• sequences mentioned in (b) are essentially the total or partial complementary sequences (in particular for the cases mentioned above).
• sequences (a) and (b) allow access not only to the murine gene from which they originate, but also to the corresponding human genes by homology.
• the invention also relates to the nucieotide sequences of the genes having a strong homology with the genes mentioned above, preferably a homology greater than 80% on the essential parts of said genes, ie in general at least 50% of the sequence, preferably the homology will be on these parts higher than 90%.
• the present invention also relates to the sequences coding for the same protein, taking into account the degeneration of the genetic code, but also for equivalent proteins, that is to say producing the same effects. , in particular proteins deleted and / or having undergone point mutations.
• sequences according to the present invention are most particularly the sequences which are induced or inhibited during cellular apoptosis, in particular those induced by p53.
• Said genes are grouped in TSAP or "Tumor Suppressor Activated Pathway" and called TSAP 1 to TSAP 8 and human TSAP 3 corresponding to IND.SEQ. 1 to 8 and 1 1 (HUMSIAH) respectively, and in TSIP or "Tumor Suppressor Inhibited Pathway” and called TSIP 1 and TSIP 2, corresponding to IND.SEQ.9 and 10.
• nucleotide sequences corresponding to the TSAP genes are sequences expressed during the process of apoptosis while when they are not expressed the process of oncogenesis continues. It is therefore interesting:
• TSIP genes can intervene in other processes than oncogenic processes; indeed, p53 is in a way the guardian of the genome gene, under these conditions the TSAP or TSIP genes are undoubtedly also involved in this control function, it is therefore all of the possible alterations in the genome which can be accountable for previous detection and therapy.
• the TSIP genes are expressed during oncogenesis and not during apoptosis, it is therefore also useful here to detect the possible TSIP anomaly and also to provide inhibition / blocking therapy.
• Replacement therapy may be carried out by gene therapy, that is to say by introducing the TSAP gene with the elements which allow its expression in vivo.
• gene therapy is to say by introducing the TSAP gene with the elements which allow its expression in vivo.
• the principles of gene therapy are known.
• the vectors can also be synthetic, that is to say mimic viral sequences, or else consist of DNA or naked RNA according to the technique developed in particular by the company VICAL.
• the present invention therefore relates to the set of vectors described above.
• the present invention also relates to cells transformed with an expression vector as described above, as well as to the protein obtainable by culturing transformed cells.
• Expression systems for producing proteins can be either eukaryotic systems such as the foregoing vectors as prokaryotic systems in bacteria cells.
• One of the advantages of the present invention is that it has demonstrated the involvement of several genes in apoptosis; thus the overexpression of one of the genes by gene therapy can, for sure; of them, lead to apoptosis only the cells in which other dysregulated genes are already expressed, that is to say malignant cells.
• the present invention also relates, as a medicament, to a compound ensuring the cellular expression of at least one of the sequences previous nuciéotides when it is induced during cellular apoptosis, in particular of the genes TSAP 1 to TSAP 8 and human TSAP 3, or on the contrary ensuring the inhibition of the cellular expression of at least one cell sequence as described above when it is inhibited during cell apoptosis, in particular TSIP 1 and TSIP 2.
• nucleotide sequences in sense or antisense strategy that is to say which can block the expression of TSIP or on the contrary, acting upstream, promoting the expression of TSAP.
• the present invention relates in particular to the use of the above drugs as an anti-cancer agent.
• the human TSAP 3 gene product (HUMSIAH) is also useful as an antiviral agent, as will appear on reading Example 2.
• the present invention therefore also relates to the use of the above drugs as an antiviral agent.
• the present invention also relates, as a diagnostic agent for determining the predisposition to cancer, all or part of the sequences according to the invention to be used as nucleotide probe or as an amplification primer, but also as an agent for diagnostic for the determination of the predisposition to cancer an antigen corresponding to all or part of the proteins encoded by the sequence according to the invention or the antibodies, in particular the corresponding monoclonal antibodies, possibly after culture.
• the diagnostic methods are known, it can be, for example, techniques of microsequencing of the variable parts after isolation and possible amplification or RFLP type detection methods or simple amplification in particular Differential techniques can, in particular, make it possible to highlight the difference between normal and abnormal TSAP or TSIP.
• the invention also relates to models implementing the above sequences.
• the human TSAP 3 gene (HUMSIAH) can be isolated, in particular by using the PCR method or other amplification methods by taking advantage of the structure of the gene. It is also possible to synthesize this gene in pieces, if necessary.
• the invention relates to an improvement to the method of Liang and Pardee (1) characterized in that in the PCR amplification a reduction in paher is carried out ("touch do wn") as described in Don et al. (2).
• C 1 mRNA also expressed by using a clone without differential expression
• C2 positive control using Cycline G and showing the induction of mRNAs corresponding to 32oC;
• MER-LTR shows the induction of this sequence at 32 ° C;
• TSAP 1 to TSAP S differential expression of the 8 mRNAs activated in the first 4 hours after induction of apoptosis;
• TSIP 1 and TSIP 2 differential expression of the 2 inhibited mRNAs in the first 4 hours after induction of apoptosis.
• lanes 1 and 2 polyA + mRNA of myeloid leukemic cells M 1 (clone S6) cultured at 37 ° C and 32 ° C respectively;
• lanes 3 and 4 polyA + mRNA of LTR6 cells cultured at 37 ° C and 32 ° C respectively; the arrow indicates the differential expression of the transcript 1.9 kb of TSAP 3 - siah 1 b of mouse;
• the arrows indicate the transcripts of 1, 9 and 2.4 kb
• A M 1 cells incubated for 4 hours at 32 ° C. and hybridized with a TSAP 3 antisense probe;
• M 1 myeloid leukemia cells (clone S6) and M 1 cells stably transfected with a temperature-sensitive mutant val 135 p53 (LTR6) (3). These cells are cultured on RPMI 1640 medium with 10% FCS at 5% CO 2 at 37 ° C. For the modification of the temperature, the cultures are placed in a second incubator at 32 ° C. For all the tests carried out in this study, the cells are tested after 12 and 24 hours for the presence of apoptosis.
• a "touch down" (2) of 10 cycles from 50 ° C to 40 ° C is carried out (94 ° C 30 seconds - 50 ° C 1 minute - 72 ° C 30 seconds ), followed by 35 cycles (94 ° C 30 seconds - 40 ° C 1 minute - 72 ° C 30 seconds) and a final extension of 5 minutes at 72 ° C
• the PCR products are separated on 6% polvacrylamide gels non denaturing (4). The gels are exposed without drying. Each differential presentation is performed by comparing M 1S6 and LTR6 at 37 ° C and after 4 hours of incubation of the two cell lines at 32 ° C.
• differentially expressed bands are cut from the gel, eluted and reamplified (1).
• PCR products are subcloned using the TA-cloning system (Invitrogen, San Diego CA) following the directions provided.
• RNA is extracted with Trizol (Life Technologies)
• PolyA + RNAs are prepared using the OhgotexdT kit (Qiagen, CA) 30 ⁇ g of total RNA or 2 ⁇ g of polyA + RNA are separated on 1% / 1 agarose ⁇ MOPS / 2% formaldehyde gel, transferred onto nylon membrane (Hybond N +, Apphgèn e, France) as described above (5).
• Northern blots are hybridized with probes labeled with P 32 on the TSAP and TSIP inserts and washed as described previously (5). To verify the induction of wild p53 function, the Northern blots are hybridized with a cyclin G probe (6).
• the blots are hybridized with a GAPDH probe.
• Different Northern blots (Clontech CA) are used under identical conditions and hybridized for control with a ⁇ -actin probe.
• the RT-PCR products for LTR6 are amplified using the following primers siah 1b: 5 'CAGTAAACCACTGAAAAACC3' and 5 ° CAAACCAAACCAAAACCAC3 '.
• the subcloned PCR product is used as a siah 1b control probe.
• Northern blots are exposed for 10 days to -80 ° C.
• the reproducibility of the results obtained by the Northern blot analyzes The blots are prepared (Bio-Rad, Hercules CA) by placing the PCR products (200 ng of Zeta-Probe Blotting Membranes, Bio-Rad, according to the manufacturer's instructions) of TSAP clones and hybridized with a P-labeled cDNA probe 32 (Superscript II Gibco-BRL, Life Technologies) corresponding to the RNA of LTR6 cells incubated at 37 ° C and then 4 hours at 32 ° C.
• the PCR product of the clone containing cyciine G is also deposited on the membranes and used as a positive control. Slot blots are exposed overnight at - 80 ° C.
• the cells are washed 3 times in a phosphate buffered saline
• RNA labeled with digoxigenin-1 1 -uredine-5 ′ -phosphate (DIG) and with biotin-1 1-UTP of TSAP 3 are used in the anaivses according to the procedure described previously (Boehringer-Mannheim).
• DIG digoxigenin-1 1 -uredine-5 ′ -phosphate
• biotin-1 1-UTP of TSAP 3 are used in the anaivses according to the procedure described previously (Boehringer-Mannheim).
• SAD-10 10 nm of anti-sheep DIG antibodies labeled with gold at 1/1000 dilution, Biocell UK. The analysis is performed using confocal laser microscopy.
• the p53 gene is, in the current state of our knowledge, the tumor suppressor which is mutated in the greatest number of cancers of very diverse origins, and the use of the temperature-sensitive mutant val-135 p53 has already shown previously, provide very important information concerning the functioning of wild-type p53 by inducing either the arrest of cell growth in G-1 phase, or the initiation of the cell death program.
• genes downstream of p53 include gadd 45, mdm 2, mck, "Mouse endogenous retrovirus” LTR, p21-waf and Cy cline G.
• the present invention has made it possible to demonstrate the existence of 1 1 genes which are differentially expressed in cells expressing p53 in its active suppressor form or in tumor cells expressing the non-active p53 gene.
• FIG. 1 shows the quantification of the hybridization signals corresponding to the differential expression of 8 of these genes which are activated at 32 ° C., that is to say in which the function of wild-type p53 is activated and therefore leads to apoptosis of the cells, these genes which are activated will be referred to below as TSAP (for Tumor Suppressor Activated Pathway), on the other hand it is noted that in two experiments 2 genes expressed at 37 ° C are partly inhibited at 32 ° C, which would imply that they are inhibited during programmed cell death, these genes have been called TSIP (for Tumor Suppressor Inhibited Pathway).
• TSAP Tumor Suppressor Activated Pathway
• TSIP 1 cDNA which is inhibited in its expression during apoptosis, it shows no homology with known genes.
• the present invention therefore also relates, as a medicament, to a compound ensuring the cellular expression of TSIP 2 intended for the treatment of Alzheimer's disease as well as as a diagnostic agent for the determination of the predisposition to Alzheimer's disease, all or part of the TSIP 2 sequence to be used as an otidic nucleic acid probe or as an amplification primer as well as an antigen corresponding to all or part of the proteins encoded by TSIP 2 or the antibodies, in particular the antibodies corresponding monoclonals, possibly after culture.
• TSAP 1 is homologous to rat phosphohpase C beta 4.
• the TSAP 1 sequence exhibits 100% identity with the PLC between nucleosides 3967 and 3985, 82% between nucleotides 3986 and 41 16 and 85% between nucleotides 4070 and 4220 (FIG. 4).
• PLC is known to be involved in the signaling pathway of tyrosine kinase receptors, and to catalyze the hydrolysis of phosphatidylinositol-4,5-biphosphate to diacyIglycerol and inositol-1,4,5-triphosphate.
• PLC is a downstream target in p53-mediated apoptosis.
• TSAP 2 shows conserved sequences (92% identity between nucleotides 259 and 299; 100% identity between nucleotides 418 and 458 and 92% identity between nucleotides 645 and 685) with the protein digested with zinc (ZFM 1) which is located in the Multiple Endocrine Neoplasia locus (MEN 1) ( Figure 5).
• ZFM 1 is a dominant autosomal disorder with a base of tumors affecting the anterior lobe of the pituitary and parathyroid glands and the cells of the pancreatic islets. It is particularly interesting to have demonstrated that both ZFM and a PLC isoenzyme are colocahsed in the same chromosomal region 1 1 q 13 containing the gene for susceptibility to MEN 1.
• the homologous regions are located on chromosome 19B. Finding that TSAP 1 and TSAP 2 are activated in response to p53 may suggest that these genes belong to a more global tumor suppression pathway and that p53 may cooperate with MEN 1.
• TSAP 3 is identical to Siah 1 b.
• This gene is the homolog in the vertebrae of the Drosophila seven in absentia (sina) gene.
• the clone described has 94% identity with the murine counterpart (nucleotides 1496 to 1634) (FIG. 6).
• TSA P 3 probe By Northern blot analysis using a TSA P 3 probe, it was possible to detect a differential expression of a messenger of 1.9 kb of this gene (FIG. 2A). This is confirmed by using a second probe corresponding to the same region of the siah 1b sequence described (FIG. 2B).
• FIG. 2C shows the tissue distribution of this gene using a TSAP 3 probe which detects both the 1.9 and 2.4 kb mRNA corresponding to the results mentioned above when a siah probe is used.
• In situ hybridization shows that TSAP 3 mRNA is induced rapidly 1 hour after induction of apoptosis (Figure 3D). His expression increases after 2 and 4 hours ( Figures 3E and 3 F) In the cells which have entered mitosis no signal is detected.
• TSAP 3 / siah 1 b is activated in the cell death program in M 1 cells induced by the p53 tumor suppressor gene.
• this gene codes for a protein digested with nuclear zinc, it could be a regulatory transcription factor which is downstream of the p53 signal.
• the results also show a direct link between genes for development in Drosophila and a major pathway for tumor suppression.
• TSAP 3 murine cDNA fragment obtained by differential analysis of mRNA
• FIG. 7 shows the cDNA and amino acid sequence of the human sina gene (TSAP 3).
• This sequence codes for a 282 amino acid piotein with a zinc digite motif C3HC4
• This protein also has analogies with proteins capable of binding to RNA
• the amino acid sequence is very conserved between Drosophila, mice and the gene human ( Figure 7).
• tissue distribution indicates that the human sina is ubiquitously expressed and codes for an mRNA of 2.3 kb and, in the placenta there is an additional transcript of 2.5 kb.
• Fluorescence by in situ hybridization (FIS H) using clones Y AC and BAC shows that the seven in absentia is localized on chromosome 16q 12-13, that is to say in a region containing the candidate tumor suppressor genes in various cancers, in particular: breast cancer (9), Wilm's tumor (10-12), Laurence-Moon-Bard et-Biedl syndrome (1 3), Beck with-Wiederman syndrome (14).
• KS cells selected from human K562 erythro-leukemia cells. While the parental K562 cells are sensitive to the cytopathic effect of the parvovirus H-1, the KS cells are resistant. These resistant cells replicate the wild type of p53 and have a suppressed phenotype both in vitro and in vivo.
• daughter cells US3 and US4 were selected from a monoclone of human U937 monocytic leukemia. These clones are resistant to the cytopathic effect of H-1 parvoviruses and show a reversion of the malignant phenotype in vivo. Analysis of surface markers for 20 cells indicates that there is no displacement in the stage of differentiation between U937 and the US clones indicating that the suppression of the malignant phenotypc is not due to a terminal differentiation.
• the sina gene is activated by the wild type p53 inducible in M 1 cells as well as in KS cells which reexpress the wild type p53.
• sina is activated in cells that become apoptotic, as shown by double labeling using a sina probe for in situ hybridization combined with a TUNEL assay.
• sina is located at the crossroads of the p53 and WAF-1 pathways.

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